Use of modified extracellular matrix proteins in diagnosis and treatment of atherosclerosis

ABSTRACT

The present invention relates to the use of fibronectin, tenascin, collagens type I, III, VI and/or VIII modified by aldehyde or by glycosylation in ELISA for detection of antibodies in plasma and serum to diagnose atherosclerosis as well as the use of induction of tolerance and active as well as passive immunization against glycosylated or aldehydemodified fibronectin, tenascin, collagen type I, III, VI and/or VIII for prevention and treatment of atherosclerosis.

PRIORITY INFORMATION

This application is a continuation of now abandoned U.S. Utilityapplication Ser. No. 11/523,366 filed Sep. 19, 2006, which is acontinuation of PCT application No. PCT/SE2005/000394, filed on Mar. 17,2005, which claims priority to Swedish Application No. 0400683-9, filedon Mar. 19, 2004, all of which are incorporated herein by reference intheir entireties.

TECHNICAL FIELD

The present invention relates to the use of certain connective tissueproteins or derivatives thereof for detection of antibodies in plasmaand serum to diagnose atherosclerosis, as well as the use of antibodiesagainst such connective tissue proteins or derivatives thereof producedby immunization or recombinant technique for detection of certainconnective tissue proteins or derivatives thereof in plasma and serum todiagnose presence of unstable and rupture-prone atherosclerotic plaques.

BACKGROUND OF THE INVENTION

Atherosclerosis is a degenerative disease of medium- and large-sizedarteries. It is the major cause of acute myocardial infarction, strokeand peripheral artery disease. The first stages of the disease arecharacterized by accumulation of cholesterol-loaded macrophages formingsmall fatty streaks on the inside of the arterial wall. Activation of achronic inflammatory process within these fatty streaks leads to theformation of raised fibromuscular plaques with various degree ofextracellular lipid and cell necrosis. In plaques with extensive celldeath and inflammation the fibrous cap covering the core of lipiddeposits becomes eroded increasing the risk for plaque rupture andthrombotic occlusion of the vessel, i.e. the major cause of acuteclinical events.

The disease is initiated by accumulation of lipoproteins, primarily LDL,in the extracellular matrix of the arterial intima. This accumulation iscaused by binding of positively charged amino acids in the LDL proteinapo B-100 to negatively charged matrix glycoproteins. The entrapped LDLaggregates and becomes oxidized in response various enzymes and oxygenradicals present in the arterial wall. The oxidation of LDL isassociated with formation of a number of highly reactive compounds suchas aldehydes, lipid peroxides and oxysterols that will causeneighbouring endothelial cells to express adhesion molecules andactivate an inflammatory response. As a result monocytes and T cellswill infiltrate the affected intima and the monocytes will differentiateinto macrophages expressing different types of scavenger receptors.These receptors effectively bind and mediate the uptake and removal ofoxidized LDL from the extracellular matrix. The macrophages willsubsequently express oxidized LDL antigens associated with HLA-DRreceptors. Recognition of these antigens by specific T cells results inactivation of an adaptive immune response against oxidized LDL. Theimportant role of this immune response in the development ofatherosclerosis is becoming increasingly recognized.

The oxidation of LDL is also associated with a degradation of apo B-100into peptide fragments that are further modified by reaction aldehydessuch as malondialdehyde (MDA) and 4-hydroxynonenal. Thesealdehyde-modified peptide fragments become major targets for the immunesystem and antibodies against MDA-modified apo B-100 fragments arefrequently encountered in human plasma. The role of these immuneresponses remains to be fully characterized but animal experimentsdemonstrating that immunization with oxidized LDL inhibits thedevelopment of atherosclerosis suggest that at least some of theseimmune responses have a protective effect.

SUMMARY OF THE PRESENT INVENTION

As discussed above it is generally believed that the oxidation of LDLmainly occurs while LDL is bound to glycosaminoglycans and otherextracellular matrix proteins in the arterial wall. However, apossibility that remains largely unexplored is that reactive aldehydesthat are generated during LDL oxidation may not only react with apoB-100 but also with the extracellular matrix proteins to which the LDLis adhered. If this happens it is likely to result in fragmentation andaldehyde-modification also of the respective matrix protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 are graphs showing the presence of antibodies againstDMA-modified extracellular matrix proteins in normal human plasma;

FIG. 3 is a graph showing inhibition of antibody binding withDMA-fibronectin, but not with native fibronectin and;

FIG. 4 includes graphs showing antibodies against MDA modified FN andfibronectin.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention in particular relates to the use of fibronectin,tenascin, collagens type I, III, VI and/or VIII modified by aldehyde orby glycosylation in ELISA for detection of antibodies in plasma andserum to diagnose atherosclerosis.

In a preferred embodiment the fibronectin, tenascin, collagens type I,III, VI and VIII have been modified by malondialdehyde.

A further preferred embodiment relates to the use of antibodies againstglycosylated or aldehyde-modified fibronectin, tenascin, collagens typeI, III, VI and VIII produced by immunization or recombinant techniquefor detection of aldehyde-modified or glycosylated fibronectin,tenascin, collagen type I, III, VI and/or VIII in plasma and serum todiagnose presence of unstable and rupture-prone atherosclerotic plaques.

A still further preferred embodiment relates to the use of labelledantibodies against glycosylated or aldehyde-modified fibronectin,tenascin, collagens type I, III, VI and/or VIII produced by immunizationor recombinant technique in imaging of atherosclerotic plaques.

A further aspect of the invention relates to the use of induction oftolerance and active as well as passive immunization againstglycosylated or aldehyde-modified fibronectin, tenascin, collagen typeI, III, VI and/or VIII for prevention and treatment of atherosclerosis.

When an aldehyde-modification and fragmentation of extracellular matrixproteins occurs in atherosclerotic plaques it is an important part ofthe disease process. Like the aldehyde-modified fragments of apo B-100also aldehyde-modified peptide sequences in extracellular matrixproteins may become targets for autoimmune responses. This will resultin recruitment of macrophage releasing matrix-degrading enzymes anderosion of plaque connective tissue and increase the risk for plaquerupture. When so, an aldehyde modification of extracellular matrixproteins is a critical factor in development of plaque rupture and acutecardiovascular events. It also points to the possibility of developingnovel therapies for atherosclerosis by manipulating these immuneresponses through passive or active immunization or by induction oftolerance depending on if the main effect of the immune response isatherogenic or atheroprotective.

The enzymatic degradation of aldehyde-modified matrix proteins is alsoassociated with a release of such degradation products into thecirculation. This is of importance because the presence of such proteinsin the circulation may act as markers of emerging plaque instability.The availability of a marker for emerging plaque instability would be ofconsiderable value in identifying patients at high risk for developmentof acute myocardial infarction and stroke. Several effective invasiveand non-invasive therapies for unstable plaques have been clinicallyestablished, However, the challenge remains to identify subjects withunstable plaques before they suffer an acute event. In subjectssuffering from an acute myocardial infarction up to 30% die beforereaching the hospital.

Results

To provide initial indirect evidence for the existence of oxidativedamages of extracellular matrix proteins in humans presence of immuneresponse against aldehyde-modified extracellular matrix proteins wasinvestigated by analyzing if antibodies against such proteins arepresent in human plasma.

The vascular extracellular matrix proteins fibronectin, tenascin,biglycan, decorin, collagen type I, III, VI and VIII were coated on thebottom of 96-well microtiter plates. The plates were subsequently washedin 0.05% Tween-20 and incubated with Superblock. Aldehyde-modificationwas achieved by incubation with 0.05M MDA for 37° C. for 3 hr. Afterwashing in 0.05% Tween-20 the wells were incubated with human plasma(pooled from 50 healthy blood donors), rinsed and incubated with goatanti-human biotinylated IgM. Detection was performed using AP-conjugatedstreptavidin and NPP substrate.

The results obtained in the ELISA analyses demonstrated presence ofantibodies against aldehyde-modified fibronectin, tenascin, collagentype I, III, VI and VIII, but not against aldehyde-modified biglycan anddecorin in human plasma (FIGS. 1 and 2). These findings demonstrate thatimmune responses against vascular extracellular matrix proteins exist inhumans. Accordingly, they also provide evidence for the existence ofoxidative damages of extracellular matrix proteins.

The antibody assay may utilise any tissue and the determinations aremade using any immuno-cyto-histochemical method, any immunoassayincluding, ELISA, Elispot, RIA and others any blotting technique,including Western blotting, Southern blotting and others, any bioassay,any tissue culture technique, RT-PCR, flow cytometry, cytometric beadarray, DNA microarray and/or proteomics.

Additional preliminary experiments were done on immune responses againstMDA modified fibronectin. It was found that preincubation of humanplasma with MDA modified fibronectin inhibited antibody-binding in theELISA demonstrating the specificity of the assay (FIG. 3).

Finally, a small pilot clinical study was performed to assess the roleof immune responses against aldehyde-modified fibronectin incardiovascular disease. The study group was recruited from the MalmöDiet Cancer Study and consisted of 26 subjects that developed an acutemyocardial infraction during a 5-year follow up period and 26 healthycontrols matched for age, sex and smoking. The level of antibodiesagainst aldehyde-modified fibronectin was determined in plasma samplesobtained at the baseline investigation. Antibodies againstaldehyde-modified fibronectin tended to be higher among those that laterdeveloped a myocardial infarction whereas the levels of antibodiesagainst native fibronectin were low an equally distributed between thetwo groups (FIG. 4).

There was also a trend for an association between antibodies againstaldehyde-modified fibronectin and carotid artery intima media thickness(an indirect measure of the severity of atherosclerosis) in the study(r=0.20, p=0.11). These observations suggest that immune responsesagainst aldehyde matrix proteins such as fibronectin may be involved inatherosclerosis and serve as markers of disease risk and severity.However, additional studies are required in order to establish thishypothesis.

For the induction of tolerance mucosal immunization is used, whereby anobtained activation of the immuno response or tolerance is dependent onthe dose of antigen and has to be tested out, as such.

To provide for an active immunization antigen extracted proteins orsynthetic peptide fragments. Theoretically recombinantly producedproteins and protein fragments can be used as well.

At passive immunization one may use either antibodies directed to wholeproteins or to peptides.

1-4. (canceled)
 5. The use of antibodies against glycosylatedfibronectin, tenascin, collagens type I, III, VI and/or VIII produced byimmunization or recombinant technique for detection of aldehyde-modifiedfibronectin, tenascin, collagen type I, III, VI and VIII in plasma andserum to diagnose presence of unstable and rupture-prone atheroscleroticplaques.
 6. The use of antibodies against aldehyde-modified fibronectin,tenascin, collagens type I, III, VI and/or VIII produced by immunizationor recombinant technique for detection of aldehyde-modified fibronectin,tenascin, collagen type I, III, VI and VIII in plasma and serum todiagnose presence of unstable and rupture-prone atherosclerotic plaques.7. The use of labeled antibodies against glycosylated fibronectin,tenascin, collagens type I, III, VI and/or VIII produced by immunizationor recombinant technique in imaging of atherosclerotic plaques.
 8. Theuse of labeled antibodies against aldehyde-modified fibronectin,tenascin, collagens type I, III, VI and/or VIII produced by immunizationor recombinant technique in imaging of atherosclerotic plaques.
 9. Theuse of induction of tolerance against glycosylated fibronectin,tenascin, collagen type I, III, VI and/or VIII for prevention andtreatment of atherosclerosis.
 10. The use of induction of toleranceagainst aldehyde-modified fibronectin, tenascin, collagen type I, III,VI and/or VIII for prevention and treatment of atherosclerosis.
 11. Theuse of active immunization against glycosylated fibronectin, tenascin,collagen type I, III, VI and/or VIII for prevention and treatment ofatherosclerosis.
 12. The use of active immunization againstaldehyde-modified fibronectin, tenascin, collagen type I, III, VI and/orVIII for prevention and treatment of atherosclerosis.
 13. The use ofpassive immunization against glycosylated fibronectin, tenascin,collagen type I, III, VI and/or VIII for prevention and treatment ofatherosclerosis.
 14. The use of passive immunization againstaldehyde-modified fibronectin, tenascin, collagen type I, III, VI and/orVIII for prevention and treatment of atherosclerosis.
 15. A method ofdetecting antibodies against aldehyde-modified fibronectin, tenacin orcollagens type I, III, VI or VII in a plasma or serum sample to diagnoseatherosclerosis, said method comprising detecting antibodies utilizingaldehyde-modified fibronectin, tenascin, collagens type I, III, VI orVIII, respectively, and wherein an increased presence of such antibodiesin the sample compared to the level of such antibodies in plasma orserum from a healthy individual indicated that the sample has beenobtained from an individual having arthrosclerosis.
 16. The methodaccording to claim 15, wherein the antibody assay is an ELISA.
 17. Themethod according to claim 15, wherein the fibronectin, tenascin,collagens type I, III, VI and VIII have been modified bymalondialdehyde.
 18. The method according to claim 15, wherein thefibronectin, tenascin, collagens type I, III, VI and VIII have beenmodified by 4-hydroxynonenal